We have been studying the molecular biology of a significant human pathogen, hepatitis C virus (HCV). Our long-term goal is to understand, at the molecular level, the precise roles of viral proteins, RNA elements, and host factors in HCV replication and pathogenesis. Studies on this enveloped positive-sense RNA virus have been (and continue to be) challenging because of its poor replication in cell cultures and the lack of a convenient animal model. Despite this, our work, together with that of other labs, has resulted in a reasonably complete picture of HCV genome structure, the biosynthesis of viral structural and nonstructural (NS) proteins, and provided assays for each of the predicted or discovered virus-encoded enzymatic activities. During the course of this work, we made two unexpected discoveries which deserve further study. First, we identified a novel 101-base RNA sequence at the 3' terminus of HCV genome RNA. This element is highly conserved among HCV genotypes suggesting an essential role in viral RNA replication, packaging, or both. We propose to characterize the secondary structure of this RNA element, to identify viral or host components which selectively bind to this structure, and to define the molecular determinants of these RNA- protein interactions. Successful development of infectious clones or other assays for HCV RNA replication and/or encapsidation will allow us to directly test the functional importance of this element. Second, we found that the 58-kDa NS5A protein, a presumed RNA replicase component and one of the two remaining NS proteins without known activities, is posttranslationally phosphorylated on serine residues. NS5A phosphorylation occurs in various cell cultures and cell-free assays and preliminary results suggest that it may be the target of one or more host kinases. We propose to identify the kinase(s) responsible for NS5A phosphorylation and assess the importance of NS5A phosphorylation in HCV replication and virus-host interactions. These studies should provide valuable new information on the HCV 3' terminal element, the NS5A phosphoprotein, and may lead to the identification of host factors required for HCV replication. Such analyses are critical for understanding each step in the virus life cycle at a level which will allow the design of protective vaccines and effective therapy for chronically infected patients.